Compositions and methods for plant transformation and regeneration

ABSTRACT

Improved compositions and methods for transformation and regeneration of plants from embryogenic callus are disclosed that include, for example: use of an intermediate-incubation medium after callus induction to increase the competence of the transformed cells for regeneration; dim light conditions during early phases of selection; use of green callus tissue as a target for microprojectile bombardment; and media with optimized levels of phytohormones and copper concentrations.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of U.S. application Ser. No.08/845,939, filed Apr. 29, 1997, which is hereby incorporated herein inits entirety by reference.

BACKGROUND OF THE INVENTION

[0002] This invention relates to compositions and methods for the invitro culture, transformation, and regeneration of plants.

[0003] Genetic improvement of various crop species by geneticengineering has sometimes been hindered because techniques for in vitroculture, transformation, and regeneration of model cultivars are lesseffective with recalcitrant commercial cultivars.

[0004] Barley (Hordeum vulgare L.) is one of the world's most importantcereal crops, closely following wheat, rice, and maize in totalproduction. Barley is used worldwide for feed, food, and maltingpurposes.

[0005] The ability to genetically engineer barley to improve itsperformance and pest-resistance or to enhance alternative uses is ofgreat importance. The practical utility of stable transformationtechnologies is largely dependent on the availability of efficientmethods for generating large numbers of fertile green plants from tissueculture materials. Procedures have been described for generating highlyembryogenic barley callus and regenerating green plants (Dale andDambrogio, 1979; Handel et al., 1985; Thomas and Scott, 1985;Goldenstein and Kronstadt, 1986; Lürz and Lörz, 1987; Wan and Lemaux,1994; Hagio et al., 1995; Rahleen, 1996). However, presently availableprocedures for producing embryogenic callus and regenerating greenplants have been of limited utility when used in transformationprocedures for commercially important barley genotypes. These procedureshave been hampered by a gradual loss of the embryogenic capacity andregenerability of callus tissue and an increase in albino(chlorophyll-deficient) plants during the prolonged periods needed toselect transformed tissue. For example, of the independently transformedcallus lines generated by one transformation procedure for the barleygenotype Golden Promise, only 51% of transformed lines give rise togreen plants and some of these lines regenerated only a small number ofgreen plants (Wan and Lemaux, 1994; Lemaux et al., 1996). When the sameprocedure was applied to the commercial barley genotypes Moravian IIIand Galena, none of the resulting transformed lines gave rise to greenplants.

[0006] There is a need, therefore, for efficient methods fortransformation and regeneration that can be used with a wide variety ofbarley genotypes, including commercially important genotypes.

SUMMARY OF THE INVENTION

[0007] We have developed improved methods and compositions for planttransformation and regeneration. The examples below detail theapplication of these methods and compositions to various barleygenotypes, including commercially important genotypes that have provendifficult or impossible to transform and regenerate by previouslyavailable methods. These improved methods, when applied to barley,result in a significantly higher regeneration frequency, reducesomaclonal variation, and improve the incidence of fertile, greentransformed plants. The methods of the present invention are not limitedto barley, however, but can be used for transformation and regenerationof other plant species.

[0008] One aspect of the present invention encompasses methods forproducing a transformed plant that include an intermediate incubationstep that improves the frequency with which transformed plants areobtained from independent transformation events. More specifically, suchmethods comprise the steps of:

[0009] (1) transforming a cell of a target plant tissue (e.g., immatureembryo, callus, microspore-derived embryo, etc.) to produce atransformed cell;

[0010] (2) culturing the transformed cell on a callus-induction medium(CIM) that includes an auxin to promote proliferation of the transformedcell and formation of a transformed callus, i.e., a callus arising fromthe initial transformation event (in some embodiments, the CIM alsocontains a low level of a cytokinin and a high level of copper);

[0011] (3) culturing the transformed callus on anintermediate-incubation medium (IIM) that includes an auxin and acytokinin to promote continued proliferation of cells arising from theinitial transformation event and formation of a regenerative structure,i.e., a multicellular structure that is competent to regenerate; and

[0012] (4) culturing the regenerative structure on a regeneration medium(RM; i.e., shooting and/or rooting medium) to produce a transformedplant.

[0013] Selection for transformed cells can begin immediately afterintroduction of DNA into a cell. Alternatively, selection can beginlater, e.g., during callus induction in order to provide sufficient timefor initial cell proliferation in the absence of the selective agent.Selection is generally maintained during the intermediate incubationstep and, depending on the selective agent, can also be maintainedduring the regeneration step.

[0014] Another aspect of the present invention encompasses optimizedplant culture media and the use of such media for plant cell and tissueculture. Such optimized media include phytohormones and copper (e.g.,cupric sulfate), which improve callus quality during initiation, promotethe regenerability of the tissue, and reduce the incidence of albinismduring the period of callus maintenance and regeneration. The media alsoincludes conventional plant nutrients and can also include a carbonsource such as maltose (which is better than sucrose for initiation ofsome species, including barley, wheat, and rice).

[0015] In preferred embodiments, the CIM includes an auxin (e.g.,2,4-dichlorophenoxyacetic acid or dicamba), for example at aconcentration of about 0.1 mg/L to about. 5.0 mg/L, preferably about 1.0mg/L to about 2.5 mg/L. The CIM can also include a cytokinin (e.g.,6-benzylaminopurine, zeatin, and kinetin), e.g., at a concentration ofabout 0.01 mg/L to about 0.5 mg/L for initial callus induction and about0.1 mg/L to about 2.0 mg/L for maintenance of callus and green tissues.

[0016] In preferred embodiments, the IIM contains an auxin, e.g., at aconcentration of about 0.1 mg/L to about 5.0 mg/L, preferably about 0.5mg/L to about 2.5 mg/L, and a cytokinin, e.g., at a concentration ofabout 0.1 mg/L to about 5.0 mg/L, preferably about 0.1 mg/L to about 2.0mg/L.

[0017] The CIM and IIM also preferably include copper, e.g., aconcentration of about 0.1 μM to about 50 μM.

[0018] Another aspect of the present invention encompasses the use ofdim light conditions during early phases of selection. Dim lightconditions allow callus to become green and reduce the incidence ofregeneration of fertile green plants, and may improve the regenerabilityof the callus tissue. Dim light conditions also permit one to screen forgreen portions of the callus (for barley, for example; yellow-greenportions for wheat), which are more likely to be regenerable. Greencallus is useful as a target plant tissue for transformation, e.g., bymicroprojectile bombardment or infection by Agrobacterium. Callus grownin dim light on a CIM develops or maintains regenerative structures andcan be maintained in this state for at least ten months for GoldenPromise, Galena, and Harrington, and at least four to six months forMorex, for example.

[0019] Another aspect of the present invention is the use ofmicroprojectile bombardment for plant transformation, wherein thebombardment is performed below 1300 psi, e.g. at 450-900 psi. Loweringthe rupture pressure and -hence the speed of the microprojectileslessens damage to the target tissue and results in less stress to thetransformed cells.

[0020] Another aspect of the present invention encompasses transformedplants and plant culture media as described herein.

[0021] The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1A shows the relative growth rate (g/g fresh weight/day) ofcallus of the barley genotype Golden Promise grown on fourteen differentmedia. (The auxin and cytokinin concentrations of the media are given inTable 1.)

[0023]FIG. 1B shows the relative growth rate (g/g fresh weight/day) ofcallus of the barley genotype Galena (B) grown on fourteen differentmedia. (The auxin and cytokinin concentrations of the media are given inTable 1.)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] We have developed improved methods for plant transformation andregeneration and compositions useful for such methods. Although thesemethods are generally applicable to barley varieties, includingrecalcitrant genotypes, they are also applicable to other plant speciesas well.

[0025] Definitions and Methods

[0026] Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art. Inaddition to the definitions of terms provided below, definitions ofcommon terms in molecular biology may also be found in Rieger et al.,1991; and Lewin, 1994.

[0027] Plant Transformation and Recreneration

[0028] “Transformed”; “Transgenic”. A cell, tissue, organ, or organisminto which a foreign nucleic acid, such as a recombinant vector, hasbeen introduced is considered “transformed” or “transgenic,” as isprogeny thereof in which the foreign nucleic acid is present.

[0029] “Foreign” nucleic acids are nucleic acids that would not normallybe present in the host cell, particularly nucleic acids that have beenmodified by recombinant DNA techniques. The term “foreign” nucleic acidsalso includes host genes that are placed under the control of a newpromoter or terminator sequence, for example, by conventionaltechniques.

[0030] Transformation by particle bombardment. Particle bombardment hasbeen employed for transformation of a number of plant species, includingbarley (see, e.g., Wan and Lemaux, 1994, and BioRad Technical Bulletin2007) and corn (see, e.g., Gordon-Kamm et al., 1990). Successfultransformation by particle bombardment requires that the target cellsare actively dividing, accessible to microprojectiles, culturable invitro, and totipotent, i.e., capable of regeneration to produce maturefertile plants.

[0031] Target tissues for microprojectile bombardment include immatureembryos, young embryogenic callus from immature embryos, microspores,microspore-derived embryos, and apical meristem tissue. We have alsofound that green callus tissues are useful targets for bombardment, asdiscussed below.

[0032] Previously, bombardment of barley tissue such as immature zygoticembryos or young callus tissue was generally carried out at 1100 psi. Wehave found that a rupture pressure under 1100 psi, preferably less than1000 psi, more preferably about 600 to 900 psi, resulted in a highercallus-induction frequency and a higher frequency of regenerativestructures in Galena, for example, possibly due to reduced damage to thetarget tissue (although Golden Promise was unaffected in its frequency).

[0033] Green Tissues as a Target for Particle Bombardment.

[0034] Barley callus tissue that is not exposed to light is movedthrough selection as rapidly as possible, since longer culture timesresult in lower regenerability and a higher incidence of albinism(Lemaux et al., 1996). We have discovered that green barley callustissue can be maintained for more than 10 months (e.g., Golden Promise,Galena, Harrington, and Salome) on an IIM (discussed in detail below)and can subsequently regenerate at high frequency when transferred to aregeneration medium. The use of green tissues as a target fortransformation by microprojectile bombardment permits long-term cultureof barley, reducing the need to maintain high-quality donor plantsthroughout the year. It may also reduce the need to backcross barleytransformants, since the green callus tissue is more highlydifferentiated than tissue that has not been exposed to light and mayhave a lower frequency of induced mutation and be less likely to exhibitsomaclonal variation. Moreover, the incidence of albinism issignificantly reduced compared to dark-grown tissue.

[0035] Other Plant Transformation Methods.

[0036] Any conventional method may be employed to transform plants,i.e., to introduce foreign DNA into a plant cell. The generation ofstable transformants and fertile transgenic plants has been achieved,for example, in a wide variety of dicotyledonous plants and in suchcereals as rice, maize, wheat, and oat by a variety of methods.

[0037] In addition to particle bombardment, conventional methods forplant cell transformation include, but are not limited to: (1)Agrobacterium-mediated transformation, (2) microinjection, (3)polyethylene glycol (PEG) procedures, (4) liposome-mediated DNA uptake,(5) electroporation, and (6) vortexing with silica fibers.

[0038] Regeneration of Transformed Plant Cells.

[0039] Transformed plant tissues are cultured on a regeneration mediumto cause differentiation of the tissue to produce a fertile transgenicplant.

[0040] It is preferable that callus-induction and plant-regeneration beaccomplished in three stages, each involving transformed cells ortissues on a medium supporting the biological events desired at eachstage: callus induction, intermediate incubation, and regeneration.

[0041] “Callus-induction medium” (CIM) preferably promotes a fast growthrate without allowing substantial differentiation of the plant tissueinto organized structures. A transformed cell arising from introductionof foreign DNA into a cell is incubated on CIM for a time sufficient forthe cell to proliferate to form sufficient callus tissue to ensure thata sufficient number of progeny cells are produced from a singletransformed cell to form numerous somatic embryos that will give rise tonumerous transformed plants when regenerated. For that reason, CIMpreferably includes an auxin (e.g., about 0.5 mg/L to about 5.0 mg/L of2,4-dichlorophepoxyacetic acid [2,4-D] or dicamba) to promote rapid celldivision. Cytokinin levels are preferably kept low for most genotypesfor initial callus induction, particularly for recalcitrant genotypes(such as the barley genotypes Galena, Morex, or Harrington), becausehigh cytokinin levels decrease the initial growth rate of the callus(high cytokinins also interfere with selection using bialaphos, althoughnot when hygromycin or G418 is used). However, a cytokinin improvescallus quality and regenerability and may reduce the incidence ofalbinism (i.e., induce the growth of more green regenerative tissues).Therefore, low levels of a cytokinin may be included in the CIM, -e.g.,6-benzylaminopurine [BAP], zeatin, kinetin, etc., preferably BAP orkinetin, at levels of about 0.01 mg/L to about 1.0 mg/L for initialcallus induction, about 0.1 mg/L to about 2.0 mg/L for callusmaintenance. The optimal level of cytokinin depends on the genotype. CIMalso preferably contains copper (about 0.1 μM to about 50 μM).

[0042] Callus tissue is divided into smaller pieces (e.g., for barley,pieces of about 3 to 5 mm are preferred) and subcultured, i.e.,transferred to fresh medium, at regular intervals to promote optimalgrowth rates. For barley, the tissue is subcultured at an interval ofabout 2-3 weeks if a low level (about 0.01 mg/L) of BAP is used andabout 3-4 weeks if a higher level of BAP is used (about 0.1 mg/L toabout 0.5 mg/L).

[0043] Preferably, the tissues are initially cultured without selection.In Example 4 below, for example, selection was not applied immediatelyafter bombardment in order to allow for the proliferation of transformedcells in the absence of dead or dying cells resulting from wounding orselection (about 1-2 weeks if immature embryos are used as a targetsource and 3-4 weeks if green tissues are used). After this period,selection is applied to select for transformed cells. Selection can beaccomplished by adding a selection agent to the culture medium for whichthe foreign DNA in transformed cells confers resistance (assuming that aselectable marker is included on the foreign DNA). Putativetransformants are identified by their faster growth on the selectivemedium relative to nontransformed-tissue. Screenable markers (e.g.,green fluorescent protein) can also be used to identify transformedtissue.

[0044] Transformed tissues preferably are maintained initially on CIM inthe dark (e.g., for about 3-4 weeks on CIM as in Example 3), thencultured under dim light conditions (for barley, approximately 10 to 30μE m⁻²s⁻¹). The use of dim light conditions has been found to reduce oreliminate the regeneration of albino barley plants (as observed in Wanand Lemaux, 1994).

[0045] For barley, embryogenic structures appear as fast-growing shiny,slightly brown-colored, nodular, compact structures. Under dim lightthese structures often appear as multiple meristem-like structures withsmall green shoots. By contrast, nontransformed tissues generally lacknodular structures and appear watery, loose and friable, or round andslow-growing. After embryogenic structures are observed in theputatively transformed tissue, the tissue is transferred to an“intermediate-incubation medium” (IIM). Incubation of the tissue on anIIM permits continued rapid growth, albeit at a slower pace than CIM.Incubation on an IIM improves the likelihood of the formation ofregenerative structures and competence for regeneration by promoting thetransition of the developmental pathway of a plant tissue from anembryogenic route to an organogenic route.

[0046] IIM suppresses the extension of shoots and can be used tomaintain and proliferate green sectors or green vegetative structuresfor long periods of time until they have reached sizes and numbersappropriate for regeneration (with barley, green regenerative tissues ofcertain genotypes can be maintained for more than ten months on DBC2medium, (the composition of which is given below), at least about eightmonths for Golden Promise, Galena and Harrington, and at least aboutfour to six months for Morex, for example).

[0047] IIM preferably includes an auxin (about 0.5 mg/L to about 2.5mg/L 2,4-D or dicamba) for continued cell proliferation. IIM preferablyalso includes high cytokinin concentrations (e.g., about 0.1 mg/L toabout 2.0 mg/L BAP) and high copper concentrations (e.g., about 0.1 μMto about 50 μM, preferably about 5 to about 30 μM. The higher cytokininconcentration reduces the rate of cell division but promotes progress tocompetence for regeneration and might reduce the incidence of albinism.

[0048] Copper concentrations in the IIM are preferably at least as highas levels in MS medium (0.1 μM, Murashige and Skoog, 1962), preferablyat least 5-fold higher, more preferably at least 10-fold, morepreferably at least 20-fold, most preferably at least 50-fold higheroptimal copper levels vary with the genotype and species. Higher copperlevels promote improved callus quality and regenerability withoutreducing callus-induction frequency or the initial callus growth rate.High copper levels may have less effect or no effect when included inregeneration medium.

[0049] The term “copper” is used herein to include any well-knownnutritional source of copper for plant culture media, e.g., cupricsulfate.

[0050] The effects of copper and BAP on the regenerability oftransformed barley tissues appear to be more than additive, i.e., thereappears to be a synergistic effect when the IIM includes both highlevels of copper and high levels of BAP.

[0051] It is desirable to generate large numbers of plants from a singleindependently transformed callus line due to transcriptional andtranslational inactivation phenomena and somaclonal variation. Incommercial cereals, for example, the number of transformants resultingfrom conventional transformation protocols has proven limiting inefforts to employ genetic engineering to achieve crop improvement.Incubation of transformed callus on an IIM prior to transfer to aregeneration medium maximizes the frequency at which individualtransformation events give rise to transformed plant lines. The use ofan intermediate incubation step increased the regeneration frequency forGolden Promise up to at least 65 percent and resulted in an increase inthe number of transformed plants produced per callus piece of up to11.4-fold.

[0052] Transformed tissue can be transferred from IIM to rooting orregeneration medium when embryogenic structures are observed (forbarley, after about 3 or-4 rounds of subculturing or after approximately9-16 weeks post-bombardment depending on the genotype and growth rate).The selection period should be longer when BAP is used in the CIM andIIM (about 3-4 months for Golden Promise and about 4-6 months forGalena).

[0053] “Regeneration medium” (RM) promotes differentiation of totipotentplant tissues into shoots, roots, and other organized structures andeventually into plantlets that can be transferred to soil. It is oftenpreferable to employ a shooting medium to promote shoot regenerationfrom embryogenic structures and a separate rooting medium to promoteroot formation. Depending upon the genotype, different levels of anauxin (e.g., 2,4-D) and a cytokinin (e.g., BAP) provide optimal results.For many barley genotypes RM contains BAP (about 0-8 mg/L) withoutauxin. However regeneration of Morex is improved by addition of auxin(2,4-D) to the RM. Conventional shooting and rooting media areconsidered regeneration media.

[0054] Any well-known regeneration medium may be used for the practiceof the methods of the present invention. For barley, FHG medium (Hunter,1988, and described in Kasha et al., 1990) is preferred.

[0055] As used herein, “plant culture medium” refers to any medium usedin the art for supporting viability and growth of a plant cell ortissue, or for growth of whole plant specimens. Such media commonlyinclude defined components including, but not limited to, macronutrientcompounds providing nutritional sources of nitrogen, phosphorus,potassium, sulfur, calcium, magnesium, and iron; micronutrients, such asboron, molybdenum, manganese, cobalt, zinc, copper, chlorine, andiodine; carbohydrates (preferably maltose for barley, although sucrosemay be better for some species); vitamins; phytohormones; selectionagents (for transformed cells or tissues, e.g., antibiotics orherbicides); and gelling agents (e.g., agar, Bactoagar, agarose,Phytagel, Gelrite, etc.); and may include undefined components,including, but not limited to: coconut milk, casein hydrolysate, yeastextract, and activated charcoal. The medium may be either solid orliquid, although solid medium is preferred.

[0056] Any conventional plant culture medium can be used as a basis forthe formulation of CIM, IIM, and RM when appropriately supplemented. Inaddition to the media discussed in the Examples below (e.g., MS mediumand FHG medium), a number of such basal plant culture media arecommercially available from Sigma (St. Louis, Mo.) and other vendors ina dry (powdered) form for reconstitution with water, for example.

[0057] Any well-known auxin or cytokinin may be used in the practice ofthe invention. Auxins include, but are not limited to, 2,4-D, dicamba,indoleacetic acid, and naphthalenacetic acid. Cytokinins include, butare not limited to, BAP, kinetin, zeatin, zeatin riboside, andN⁶-(2-isopentenyl) adenine (2iP). A particular genotype or species mayrespond optimally to a specific phytohormone, as noted in the Examplesbelow.

[0058] Albinism. Albinism is a common problem in barley tissue culture(Kott and Kasha, 1984; Kasha et al., 1990; Jähne et al., 1991). Albinismis influenced by a number of factors, including genetic background(Foroughi-Wehr et al, 1982), physiological state of the donor plants(Goldenstein and Kronstadt, 1986), exposure to bialaphos (Wan andLemaux, 1994), length of time in culture (Bregitzer et al., 1995), andculture conditions (Kao et al., 1991).

[0059] Wan and Lemaux (1994) reported that, of 91 transgenic calluslines generated by particle bombardment of various target tissues, 36lines yielded green plants and 41 yielded only albino plants. Lemaux etal (1996) reported that, of 73 transgenic callus lines generated byparticle bombardment, 37 lines yielded green plants and 20 yielded onlyalbino plants.

[0060] The improved methods discussed herein significantly reduce theincidence of albinism below levels reported previously. Preferably, thepercentage of putative transformation events that regenerate to producegreen transformed barley plants (and not albino plants), i.e., thenumber of transformation events yielding green plants divided by thetotal number of transformation events yielding green and albinoplants×100 percent, is at least about 60 percent, preferably at leastabout 75 percent, and most preferably at least about 90 percent.

[0061] The methods described herein also reduce problems associated withinduced heritable mutation and somaclonal variation that can result fromlong-term maintenance of plant tissue in culture.

[0062] “Plant”. The term “plant” encompasses transformed plants, progenyof such transformed plants, and parts of plants, including reproductiveunits of a plant, fruit, flowers, seeds, etc. The transformation methodsand compositions of the present invention, is applicable to variousbarley genotypes (e.g., Morex, Harrington, Crystal, Stander, MoravianIII, Galena, Salome, Steptoe, Klages, Baronesse, etc.) as well as toother species of monocotyledonous plants (e.g., wheat, corn, rice,etc.), or dicotyledonous plants (e.g., tomato, potato, soybean; cotton,tobacco, etc.).

[0063] A “reproductive unit” of a plant is any totipotent part or tissueof the plant from which one can obtain progeny of the plant, including,for example, seeds, cuttings, tubers, buds, bulbs, somatic embryos,microspores, cultured cells (e.g., callus or suspension cultures), etc.

[0064] Nucleic Acids

[0065] “Isolated”. An “isolated” nucleic acid is one that has beensubstantially separated or purified away from other nucleic acidsequences in the cell of the organism in which the nucleic acidnaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA. The term also embraces recombinant nucleic acids and chemicallysynthesized nucleic acids.

[0066] “Operably Linked”. Nucleic acids can be expressed in plants orplant cells under the control of an operably linked promoter that iscapable of driving expression in a cell of a particular plant. A firstnucleic-acid sequence is “operably” linked with a second nucleic-acidsequence when the first nucleic-acid sequence is placed in a functionalrelationship with the second nucleic-acid sequence. For instance, apromoter is operably linked to a coding sequence if the promoter affectsthe transcription or expression of the coding sequence. Generally,operably linked DNA sequences are contiguous and, where necessary, tojoin two protein coding regions to produce a hybrid protein.

[0067] “Recombinant”. A “recombinant” nucleic acid is made by anartificial combination of two otherwise separated segments of sequence,e.g., by chemical synthesis or by the manipulation of isolated segmentsof nucleic acids by conventional genetic engineering techniques.

[0068] “Vectors, Transformation, Host cells”. Nucleic acids can beincorporated into recombinant nucleic-acid constructs, typically DNAconstructs, capable of being introduced into and replicating in a hostcell. Such a construct preferably is a vector that includes sequencesthat are capable of transcription and translation of apolypeptide-encoding sequence in a given host cell (and may include areplication system, although direct DNA introduction methodsconventionally used for monocot transformation do not require this).

[0069] For the practice of the present invention, conventionalcompositions and methods for preparing, and using vectors and host cellsare employed, as discussed, inter alia, in Sambrook et. al., 1989, orAusubel et al.,. 1992.

[0070] A number of vectors suitable for stable transformation of plantcells or for the establishment of transgenic plants have been describedin, e.g., Pouwels et al., 1987, Weissbach and Weissbach, 1989, andGelvin et al., 1990. Typically, plant expression vectors include, forexample, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 31 regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally-or developmentally-regulated, or cell- ortissue-, specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

[0071] Examples of constitutive plant promoters useful for expressinggenes in plant cells include, but are not limited to, the cauliflowermosaic virus (CaMV) 35S promoter, maize ubiquitin (Ubi-1) promoter, riceactin (Act) promoter, nopaline synthase promoter, and the octopinesynthase promoter. A variety of plant gene promoters that are regulatedin response to environmental, hormonal, chemical, and/or developmentalsignals also can be used for expression of foreign genes in plant cells,including promoters regulated by heat (e.g., heat shock promoters);light (e.g., pea rbcS-3A or maize rbcS promoters or chlorphylla/b-binding protein promoter); phytohormones, such as abscisic acid;wounding (e.g., wunI); anaerobiosis (e.g., Adh), and chemicals such asmethyl jasmonate, salicylic acid, or safeners. It may also beadvantageous to employ well-known organ-specific promoters such asendosperm-, embryo-, root-, phloem-, or trichome-specific promoters, forexample.

[0072] Plant expression vectors optionally include RNA processingsignals, e.g., introns, which may be positioned upstream or downstreamof a polypeptide-encoding sequence in the transgene. In addition, theexpression vectors may also include additional regulatory sequences fromthe 3′-untranslated region of plant genes, e.g., a 3′ terminator regionto increase mRNA stability of the mRNA, such as the PI-II terminatorregion of potato or the octopine or nopaline synthase 3′ terminatorregions.

[0073] Such vectors also generally include one or more dominantselectable marker genes, including genes encoding antibiotic resistance(e.g., resistance to hygromycin, kanamycin, bleomycin, G418,streptomycin, paromomycin, or spectinomycin) and herbicide-resistancegenes (e.g., resistance to phosphinothricin acetyltransferase orglyphosate) to facilitate manipulation in bacterial systems and toselect for transformed plant cells.

[0074] Screenable markers are also used for plant cell transformation,including color markers such as genes encoding β-glucuronidase (gus) oranthocyanin production, or fluorescent markers such as genes encodingluciferase or green fluorescence protein (GFP).

[0075] The invention will be better understood by reference to thefollowing Examples, which are intended to merely illustrate the bestmode now known for practicing the invention. The scope of the inventionis not to be considered limited thereto.

EXAMPLES Example 1

[0076] Improvement of Callus Quality and Regenerability in BarleyGenotypes Golden Promise and Galena

[0077] Materials and Methods

[0078] Plant Material. Donor plants for immature embryos were grown insoil under controlled conditions in growth chambers as described (Wanand Lemaux, 1994; Lemaux et al., 1996).

[0079] As noted in other Examples below, plants were grown in agreenhouse (immature embryos grown in the growth chambers are preferredfor green tissue culture). The greenhouse had supplementary lightingproviding a 14-h photoperiod with temperatures of 15 to 18° C.Supplemental 1000-watt metal-halide lights were engaged when the lightlevel in the greenhouse was less than 1000 μE m⁻² s⁻¹. Roof shadescovered the roof when outside light levels rose above 7000 μE m⁻² s⁻¹.

[0080] Spring cultivars of barley (Hordeum vulgare L.) Golden Promiseand Galena were used as donor plants. Galena seed was obtained from B.Treat, Coors Brewing Company, Golden, CO. Golden Promise seed wasobtained from P. Bregitzer, USDA-ARS Small Grains Germplasm Center,Aberdeen, Id.

[0081] “Media”. Callus-induction medium (CIM) is MS medium (Murashigeand Skoog, 1962) supplemented with 30 g/L maltose, 1.0 mg/L thiamine-HC1, 0.25 g/L myo-inositol, 1.0 g/L casein hydrolysate, 0.69 g/L proline,and solidified with 3.5 g/L Phytagel (Sigma, St. Louis, Mo.). CIM wassupplemented with fourteen different combinations of two auxins (dicambaand 2,4-dichlorophenoxyacetic acid [2,4-D]) and two cytokinins(6-benzylaminopurine [BAP] and zeatin) as shown in Table 1, and thesupplemented medium was tested for callus induction, callus quality,growth rate, and regenerability.

[0082] Regeneration medium (RM) is FHG medium (Hunter, 1988; Kasha etal., 1990), a modified MS medium with lower NH₄NO₃ and high glutamine,supplemented with 1 mg/L BAP and solidified with 3.0 g/L Phytagel. Thecomposition of FHG medium is 165 mg/L NH₄NO₃ 1.90 g/L KNO₃, 440 mg/LCaCl₂.2H₂O, 370 mg/L MgSO₄.7H₂O, 170 mg/L KH₂PO₄, 16.9 mg/L MnSO₄.H₂O,8.6 mg/L ZnSO₄.7H₂O, 6.2 mg/L H₃BO₃, 0.83 mg/L KI, 0.25 mg/LNa₂MoO₄.2H₂O, 25 μg/L CuSO₄.5H₂O, 25 μg/L CoCl₂.6H₂O, 0.4 mg/Lthiamine-HCl, 100 mg/L inositol, 730 mg/L glutamine, 62 g/L maltose,27.8 mg/L FeSO₄.7H₂O, 33.5 mg/L Na₂EDTA, 1.0 mg/L BAP, 3 g/L Phytagel,pH 5.6.

[0083] “Callus induction and scoring”. Immature embryos (about 1.5-2.5mm in size) were taken from spikes approximately three months in agethat were surface-sterilized in 20% (v/v) bleach (5.25% sodiumhypochlorite) for 7 min, washed 5 min with sterile water three times,bisected longitudinally, and placed on CIM. Ten half-embryos were testedon CIM supplemented with each of the fourteen phytohormone combinations;each treatment had three replicates. (Whole embryos can also be used.)Callus-induction frequency was measured by counting numbers of halfembryos undergoing callus induction under a light microscope 2 to 3weeks after initial culturing.

[0084] Two embryo sizes were tested: small (0.5-1.5 mm) and large(1.5-2.0 mm). Golden Promise is good in callus induction with bothsmall- and large-sized embryos, but callus induction is very poor withsmall-sized embryos of Galena.

[0085] “Callus growth rate”. For determining callus growth rates, tenhalf embryos were placed scutellum-side down on a Petri dish containingeach medium; each treatment had three replicates. Every 2 to 3 weeks,callus pieces were weighed and the growth rate was determined byweighing the plate containing the callus pieces before transfer (WI) andafter transfer of all tissue (W₂). The relative growth of the callus wascalculated as the change in weight (W) of the callus (W=W₁−W₂), dividedby the weight of the tissues originally plated (W₁) and the number ofculture days (g/g fresh weight/day). From the third transfer, three ofthe highest-quality pieces, rather than all calli from each embryo, weretransferred onto fresh medium. All calli that were not transferred wereremoved from the plate to obtain W₂.

[0086] “Callus quality”. Callus quality (morphology and color) wasassessed microscopically 2 to 3 weeks after initial callus induction.For morphology, a score of ++++(highest quality) was given to shiny,compact, nodular callus; a score of +(lowest quality) was given to soft,friable callus. Color was judged from slightly brown-colored callus(++++) to white (+).

[0087] “Regeneration”. To test regeneration, ten pieces ofhighest-quality callus (8 to 11 mg per piece) from each treatment weretransferred to RM in three replicates at varying times during theculture period. Dishes were placed at 24±1° C. under fluorescent lights(45 to 55 μE m⁻² s⁻¹, 16 h light/8 h dark). The number of shoots percallus piece was counted about 22-25 days after transfer. (One or moreleaves arising from the same base of green tissue was considered as oneshoot.)

[0088] Results

[0089] Transduction frequency, relative growth rate, and qualitativeappearance of callus. To examine the effects of different concentrationsand types of auxins and cytokinins on callus-induction frequency,quality, and relative growth rate, 14 different media were tested (Table1, left two columns). On most media, callus-induction frequencies werenot statistically different for Golden Promise and Galena; dicamba and2,4-D alone and dicamba with zeatin at all concentrations resulted innearly 100% induction frequencies for both genotypes. Golden Promise hada significantly higher callus-induction frequency than Galena on 3 ofthe 14 media tested: dicamba+0.1 mg/L BAP, dicamba+0.5 mg/L BAP, and2,4-D+0.5 mg/L BAP. Galena had a significantly higher callus-inductionfrequency than Golden Promise on only one medium, 2,4-D+0.01 mg/Lzeatin. With Galena, higher levels of BAP in combination with 2,4-D, or,more significantly, in combination with dicamba, led to lowercallus-induction frequencies.

[0090] Callus induction from Golden Promise embryos occurred over mostof the surface area of the scutellum, while Galena callus was producedfrom a much smaller area of the embryo.

[0091] Color assessments of the two genotypes on the same medium wereidentical. However, in general, callus morphology of Golden Promise wasbetter than that of Galena on nearly all media tested (Table 1). Certaintrends in morphology were seen for both genotypes. First, culturing onmedium containing BAP in combination with either 2,4-D or dicambaproduced a better callus morphology than culturing on medium containingzeatin with either 2,4-D-or dicamba (Table 1). Second, callus color inboth genotypes was dramatically affected by the type of cytokinin (Table1). Increasing BAP levels (with either auxin) led to the formation ofmore slightly brown-colored callus, whereas zeatin at all levels (witheither auxin) led to the formation of poor quality, white callus (Table1). Third, medium containing higher concentrations of BAP (0.1 to 0.5mg/L with 2,4-D) appeared to support the production of higher qualitycallus (morphology and color) than did the lower concentration of BAP(0.01 mg/L) with both genotypes (Table 1).

[0092] In the first growth period, determination of the growth rate wascomplicated by the rapid increase in fresh weight of the startingmaterial due to imbibition of the embryo. By the third transfer, therelative growth rate increased rapidly, reaching its maximum (FIG. 1).Growth rates dropped significantly after the fourth growth period. Forboth genotypes, growth rates on media containing BAP were generallyslower than in the absence of BAP or in the presence of zeatin. GoldenPromise appeared to grow faster than Galena on media containing dicambaplus BAP and 2,4-D+/−BAP. Both genotypes grew faster on mediumcontaining 2,4-D plus BAP than on medium containing dicamba plus BAP(except for Galena at 0.5 mg/L BAP). There appeared to be littlevariation between genotypes in growth rate on medium containing 2,4-D ordicamba in combination with zeatin. The use of low concentrations ofzeatin (0.01 or 0.1 mg/L) in combination with dicamba or 2,4-D did notappear to inhibit the callus growth rate of Golden Promise relative togrowth on dicamba or 2,4-D alone, and the combination of lowconcentrations of zeatin with 2,4-D seemed to increase the callus growthrate of Galena up to the fourth growth period relative to 2,4-D alone(FIG. 1).

[0093] Plant regeneration. Calli of Golden Promise and Galena grown onthe fourteen different media were tested for their ability to regenerateplants. In general, Golden Promise produced a higher number of greencalli (NC) and green shoots (NS) per 10 initial callus pieces than didGalena at most time points on most media (compare Tables 2 and 3). Inaddition, Galena callus appeared to lose regenerability at a faster ratethan Golden Promise except on callus-induction media containing BAP incombination with 2,4-D, in which case Galena responded more favorablythan Golden Promise at all levels of BAP.

[0094] For Golden Promise (Table 2), through the fifth transfer alltreatments produced comparable numbers of green calli, while 2,4-D plus0.01 and 0.5 mg/L BAP appeared to yield the highest numbers of shoots.In most cases the number of shoots and green calli decreaseddramatically after either the fifth or seventh transfers. One of themost dramatic losses was at the seventh transfer with the use of dicambaalone, where no green calli were observed. Few media supported theregeneration of plants at the ninth transfer. Only dicamba and 2,4-Dplus 0.1 mg/L BAP and 2,4-D plus 0.5 mg/L zeatin supported long-termshoot regenerability in Golden Promise.

[0095] For Galena (Table 3), on medium-containing either (1) dicamba or2,4-D with zeatin or (2) dicamba in combination with BAP, the ability togenerate green shoots was lost more rapidly than with Golden Promise.The only media supporting long-term maintenance of greening andregeneration of plants (7th transfer and beyond) was 2,4-D plus BAP atall levels. Media containing 0.1 mg/L BAP appeared optimal at the latesttime point and supported a faster callus growth rate than dicamba plus acomparable level of BAP (FIG. 1).

[0096] For both genotypes, medium containing BAP in combination with2,4-D (and to a lesser extent dicamba) supported the development ofmultiple shoots from the shiny, compact callus tissues (Tables 1-3),while few or no shoots developed on a medium containing 2,4-D alone.

[0097] Discussion

[0098] In these experiments, medium composition and phytohormone typesand levels were important factors in determining tissue cultureresponses. Certain generalizations can be made regarding the effects ofdifferent cytokinins on the properties of proliferated callus. Althoughmedium containing zeatin appeared to support faster growth rates, mediumcontaining zeatin (plus 2,4-D or dicamba) also produced lower quality(soft, light-colored) callus compared to medium containing BAP (plus2,4-D or dicamba) (Table 1).

[0099] The detrimental effects of zeatin can also be seen by comparingthe regenerative potential of calli from both genotypes grown on mediumincluding either BAP or zeatin. Calli grown on medium containing zeatin(from 0.01 to 0.5 mg/L) were less regenerative than calli grown onmedium containing BAP and regenerated on the same RM (Tables 2 and 3).This finding is in contrast to that of Lürz and Lörz, (Theor. Appl.Genet., 75:16-25, 1987), who showed that, in combination with IAA,zeatin and zeatin riboside (0.05 mg/L) increased the frequency ofregeneration, of somatic.embryos from the barley genotypes GoldenPromise and Dissa, while higher levels reduced regeneration. Othercytokinins, such as BAP, kinetin, and 2iP, were shown to cause callusbrowning and necrosis of somatic embryos. Media containing IAA andzeatin has also been shown to improve regenerablity of immatureembryo-derived callus of Hordeum spontaneum and H. bulbosum (Breimann,Plant Cell Rep., 4:161-163, 1985). The fact that we did not observe apositive effect of zeatin on the tissue culture response of GoldenPromise and Galena may result from the particular barley genotypes, thedifferent auxins (dicamba and 2,4-D) we employed, or other modificationsin our culturing procedures.

[0100] In contrast to zeatin, the addition of BAP to 2,4-D-containingmedum decreased the growth of the soft, friable callus and increased thefrequency of embryogenic, shiny, compact and slightly brown-coloredcallus that was more highly regenerative (Table 1). In many cases, calligrown on media containing low concentrations of BAP (0.01 or 0.1 mg/L)in combination with 2,4-D yielded the largest numbers of regeneratedshoots for a particular genotype; with Galena, 2,4-D+BAP prolonged theregeneration period for green plants. The auxin 2,4-D is most commonlyused for embryogenic callus formation in cereal crops, but the additionof cytokinin to 2,4-D can be significant, depending on plant species andgenotypes (reviewed by Bhaskaran and Smith, Crop Sci., 30:1328-1336,1990). Recently, multiple shoots were differentiated from excised shootapical meristems in maize (Zhong et al., Planta, 187:483-489, 1992) andoat (Zhang et al, J. Plant Physiol., 148:667-671, 1996) cultured on BAPand 2,4-D. This effect of BAP on shoot regeneration is also consistentwith previous observations on Kentucky bluegrass (Griffin and Dibble,Plant Cell Rep., 14:721-724, 1995) and creeping bentgrass (Zhong et al.,Plant Cell Rep., 10:453-456, 1991), where higher frequencies of shootregeneration from seed-derived callus were achieved when auxin (dicambaor 2,4-D) and BAP were substituted for auxin alone.

[0101] In our study the positive effect of BAP in combination with 2,4-Dwas also reflected in callus quality (Table 1). Shiny, compact andslightly brown-colored callus produced green plants. Compact,light-colored callus was regenerative but generally produced albinoplants. Soft, friable callus was not regenerative. For both GoldenPromise and Galena, the addition of BAP (in combination with either2,4-D or dicamba) decreased the growth of the soft, friable, whitecallus and increased the proportion of compact, slightly brown-coloredregenerative callus relative to no cytokinin or comparableconcentrations of zeatin (Table 1).

[0102] Golden Promise calli grown on medium containing 0.01 mg/L BAP incombination with 2,4-D regenerated almost the largest (fourth/seventhtransfers) or equivalent (fifth) numbers of green shoots relative to theother media (Table 2). Calli grown on medium containing 0.1 mg/L BAPwith 2,4-D produced fewer (and shorter) green shoots than media with0.01 mg/L at all but the third and ninth transfers (Table 2). ForGalena, growth on medium containing 2,4-D with 0.1 mg/L of BAP producedcallus that yielded the most green plants at all transfer times exceptthe third (Table 3). When calli with small, green, compact shoots weretransferred for the second time from medium containing 2,4-D and 0.1mg/L BAP onto fresh regeneration medium, more tissue containing multipleshoots was seen than when 0.01 mg/L BAP was used. It is possible thatthe BAP-containing medium caused the callus tissue to proliferate forprolonged periods in a regenerable state.

[0103] The negative effects of the length of time in culture onregenerative potential is also documented in this study. On all media,immature embryos of Golden Promise produced fast-growing., embryogeniccallus that gave rise to green plants at high frequencies for periods upto two months (fifth transfer) after initial callus induction (Table 1and 2; FIG. 1). After the fifth transfer, Golden Promise calli began tolose regenerative potential (Table 2). Galena lost regenerability muchmore rapidly than Golden Promise on all media tested, except for mediacontaining 2,4-D plus BAP (Table 3), with regenerability declining onmost media after the fourth transfer. Therefore, longer periods ofculturing in the dark led to lower total numbers of regenerated greenplants from both Golden Promise and Galena (Tables 2 and 3), with thelosses in Galena being more marked.

[0104] Culture time also appeared to affect albinism. There was asmaller number of green calli in Galena cultures relative to GoldenPromise at later time points (seventh, ninth) on most media (Tables 2and 3). Some albino plants were produced from Golden Promise at latertransfer times. However, when cultured on the same medium for the samelength of time, Galena produced larger numbers of albino plants. Thepropensity of Galena toward albinism is also supported by data collectedduring regeneration tests of 1-month-old Golden Promise and Galena calligrown on 2,4-D (2.5 mg/L) in combination with BAP (0.1 mg/L). From thismaterial, 70 to 80%; of the GP cells became green under dim lightconditions (10 to 20 μE m⁻² s⁻¹), whereas less than 20% of the cellsfrom a comparably aged Galena culture had greening potential.

[0105] Thus, the length of time in culture and genotypic differenceshave dramatic effects on albinism and hence on the ability to regenerategreen plants.

[0106] Embryo size is another important factor affectingcallus-induction frequencies.

[0107] Optimal embryo size varies with genotype. Using embryo sizeslarger than 2.5 mm from both genotypes resulted in low callus-inductionfrequencies. Galena embryos from 0.5 to 1.2 mm in size had very lowcallus-induction frequencies (<20%) while Golden Promise embryos of thesame size had a frequency of over 90%. The highest callus-inductionfrequencies with Golden Promise were associated with calli from 0.5 to2.0 mm in size, while the optimal size for Galena was 1.5 to 2.0 mm. Theeffect of size on callus-induction frequency is likely due to theeffects of the exogenously applied hormones on the developmentalcascades that are triggered in a particular sized, immature embryo andthe developmental flexibility of the particular genotype.

[0108] The frequency of induction, quality, and regenerability of callusin barley are influenced by a variety of factors, such as mediacomposition (Bregitzer, 1992; Dahleen, 1995; Handel et a!, 1985; Lürzand Lörz, 1987), phytohormones (Hagio et al., 1995; Ziauddin and Kasha,1990; Lürz and Lörz, 1987), length of time in culture (Lürz and Lörz,1987; Bregitzer et a!, 1995), embryo size (Baillie et: al., 1993;Ziauddin and Kasha, 1990; Dale and Dambrogio, 1979), and genotype(Dahleen, 1996; Baillie et a!, 1993; Bregitzer, 1992; Lürz and Lörz,1987; Goldenstein and Kronstadt, 1986; Handel et al., 1985). We haveconfirmed and expanded these observations to the transformable barleycultivar Golden Promise and a recalcitrant commercial barley variety,Galena.

[0109] Using a previously published transformation protocol thatemployed a medium containing 2.5 mg/L dicamba and no cytokinin (Wan andLemaux, 1994), we obtained large numbers of transformed callus lineswith Galena, but all lines yielded only albino plants. We haveidentified optimal combinations and levels of auxin and cytokinin forproduction of the highest quality, regenerative callus of Golden Promiseand Galena during prolonged tissue culture periods. For both genotypes,2,4-D in combination with BAP (between about 0.01 and about 0.1 mg/L)was found to be optimal for prolonging regenerability and producing thehighest numbers of green calli and shoots. These phytohormone conditionscan be adjusted for optimal results with other barley genotypes and forother plant species as well. TABLE 1 Callus-Induction Frequency andQualitative Appearance of Golden Promise and Galena Auxin Conc.Cytokinin Callus Induction Frequency (mg/L) conc. (mg/L) (%)^(a) CallusMorphology^(d) Callus Color^(e) Dicamba BAP GP^(b) GAL^(c) GP GAL GP GAL(1) 2.5 0 100 ± 0  100 ± 0  +++(+) + ++ + (2) 2.5 0.01 96.3 ± 5.2 92.6 ±5.2 +++ +(+) ++ ++ (3) 2.5 0.1 88.9 ± 0    71.3 ± 12.4 +++ ++ ++ ++ (4)2.5 0.5 92.6 ± 5.2 54.2 ± 5.9 +++ +++ +++ +++ 2,4-D (5) 2.5 0 92.6 ± 5.2100 ± 0  +++ +(+) ++ ++ (6) 2.5 0.01 100 ± 0  96.3 ± 5.2 +++(+) ++ ++++++ (7) 2.5 0.1 84.3 ± 6.6 84.3 ± 6.6 ++++ +++ ++++ ++++ (8) 2.5 0.592.6 ± 5.2 75.0 ± 0   ++++ +++ ++++ ++++ Dicamba Zeatin (9) 2.5 0.01 100± 0  96.3 ± 5.2 ++ + + + (10) 2.5 0.1 100 ± 0  100 ± 0  +++ ++ + + (11)2.5 0.5 100 ± 0  100 ± 0  ++ + + + 2,4-D (12) 2.5 0.01 79.6 ± 6.6 100 ±0  ++ + + + (13) 2.5 0.1  88.0 ± 10.2 88.9 ± 0   +(+) +(+) + + (14) 2.50.5 100 ± 0  100 ± 0  ++ + + +

[0110] TABLE 2 Regeneration of Golden Promise Calli Grown on CIM withDifferent Combinations of Auxins and Cytokinins Auxin Con. Cytokininconc. 3^(rd) Transfer 4^(th) Transfer 5^(th) Transfer 7^(th) Transfer9^(th) Transfer (mg/ml) (mg/ml) NS^(a) NC^(b) NS NC NS NC NS NC NS NCDicamba BAP 2.5 0 189 10 134 10 122 7 0 0 0 0 2.5 0.01 186 10 190 10 16310 5 1 0 0 2.5 0.1 134 10 90 10 138 10 68 7 5 2 2.5 0.5 88 10 100 10 6210 26 4 0 1 2,4-D 2.5 0 140 10 140 9 91 6 37 4 0 0 2.5 0.01 146 10 17510 216 10 51 9 0 0 2.5 0.1 213 10 120 9 85 7 17 4 7 7 2.5 0.5 139 10 12710 219 10 22 6 0 0 Dicamba Zeatin 2.5 0.01 204 10 138 10 87 6 44 3 0 02.5 0.1 186 9 120 10 94 8 0 1 0 0 2.5 0.5 114 10 89 10 139 8 34 7 0 02,4-D 2.5 0.01 165 10 125 10 90 8 81 7 0 0 2.5 0.1 87 10 121 8 88 6 41 40 0 2.5 0.5 105 10 141 10 56 7 52 6 2 3

[0111] TABLE 3 Regeneration of Galena Calli Grown on CIM with DifferentCombinations of Auxins and Cytokinins Auxin Con. Cytokinin conc. 3^(rd)Transfer 4^(th) Transfer 5^(th) Transfer 7^(th) Transfer 9^(th) Transfer(mg/ml (mg/ml) NS^(a) NC^(b) NS NC NS NC NS NC NS NC Dicamba BAP 2.5 0.60 7 63 9 15 2 0 0 0 0 2.5 0.01 54 7 52 9 0 0 0 0 0 0 2.5 0.1 22 9 31 101 2 0 0 0 0 2.5 0.5 22 8 19 4 43 7 0 6 0 0 2,4-D 2.5 0 2 1 4 1 27 7 0 00 0 2.5 0.01 46 8 22 7 24 6 11 1 13 1 2.5 0.1 18 10 105 9 65 7 24 2 26 42.5 0.5 30 6 57 10 39 5 13 5 6 6 Dicamba Zeatin 2.5 0.01 36 9 68 8 0 100 0 0 0 2.5 0.1 39 7 12 3 0 10 0 0 0 0 2.5 0.5 7 7 5 2 0 10 0 0 0 02,4-D 2.5 0.01 19 3 42 5 28 5 0 0 0 0 2.5 0.1 36 5 25 5 0 10 0 0 1 0 2.50.5 6 2 4 2 4 8 0 0 0 0

Example 2

[0112] High-Frequency Plant Regeneration from Transgenic andNontransgenic Callus Tissues of Barley

[0113] Materials and Methods.

[0114] Callus induction and maintenance. Callus induction was performedas described above using CIM with 2.5 mg/L of 2,4-D or dicamba (nocytokinin). After incubation for three weeks at 24+1° C. in the dark,callus was cut into small pieces (about 3 to 4 mm), then maintained onthe same medium with subculturing at three-week intervals.

[0115] Plasmids. Plasmid ppGlbGus-6 (Liu, 1994) contains the uidA (gus)reporter gene under the control of the maize embryo-specific globulin(Glbl) promoter (containing 1.38 kb upstream of the transcription startsite) and terminated by the Agrobacterium tumefaciens nopaline synthase3′ polyadenylation signal (nos). Plasmid pdGlbGUS-6 was constructed by(1) digesting ppGlblGUS with EcoRI to obtain a 2.54-kb fragmentcontaining 0.37-kb of the globulin promoter, uidA reporter gene and nosterminator, and (2) ligating the 2.54-kb fragment into the vector pUC19.Plasmid pAHC20 contains the bar gene from Streptomyces hygroscopicusunder the control of the maize ubiquitin Ubil promoter and first intron(Christensen and Quail, 1996) and followed by the 3′-untranslated regionand nos.

[0116] Micro-projectile bombardment and transformation. Barleytransformation via microparticle bombardment was carried out asdescribed (Wan and Lemaux, 1994).

[0117] Recreneration via an intermediate-incubation step. Ten pieces oftwo-month-old nontransgenic calli grown on CIM supplemented with either2,4-D or dicamba in the dark were transferred to RM either directly orfollowing incubation on an IIM.

[0118] Two different IIM were used, DBC2 and DBC3. DBC2 medium is CIMcontaining 2.5 mg/L 2,4-D, 0.1 mg/L BAP, and 5.0 μM copper (Cupricsulfate). DBC3 medium is CIM containing 1.0 mg/L 2,4-D, 0.5 mg/L BAP,and 5.0 FM copper. After growing calli on these media for 3-4 weeksunder dim light conditions (20 to 30 μE m⁻² s⁻¹; 16 h light/8 h dark),the numbers of calli producing green sectors or green regenerativestructures were counted. Green sectors and small green regenerativestructures were then transferred to fresh RM and grown under higherlight intensity (45-55 μE m⁻² s⁻¹). After 3-4 weeks, the numbers ofgreen shoots per callus piece were counted. For regeneration oftransgenic callus lines, seven to ten pieces of transgenic calli wereeither transferred directly to each medium (containing 4-5 mg/Lbialaphos) or transferred after an incubation on an IIM, then grownunder the same conditions as described above for nontransgenic calli.Each treatment included four replicates of the regeneration test fornontransgenic calli but one replicate for transgenic calli.

[0119] Results

[0120] Transgenic calli and nontransgenic calli grown on the CIM withand without bialaphos, respectively, were transferred onto RM eitherdirectly or after incubation on an IIM. There was no significantdifference among treatments in numbers of nontransgenic and transgeniccalli of Golden Promise producing green sectors 3-4 weeks after transfer(Tables 4 and 6). Multiple green shoots were induced from-bothtransgenic and nontransgenic calli when either DBC2 or DBC3 was used asan IIM. Incubation on an IIM resulted in multiple green structures from2,4-D and BAP and even more structures from the treatment includingelevated levels of copper. Calli on either DBC2 or DBC3 formed multiplegreen shoots from the meristem-like structures; no albino plants wereobserved. Most of the green sectors that arose directly on RM without anintermediate incubation step regenerated fewer than two shoots per greensector, while green sectors grown on an IIM produced 2-5 shoots pergreen sector (Table 4). CIM containing 2,4-D was better in green-shootregeneration than callus from medium containing dicamba (Table 4). Thefrequency of shoot regeneration was increased 5.6-fold to 6.4-fold fornontransgenic calli initiated and maintained on BCI-DM (barleycallus-induction medium [Wan and Lemaux, 1994] containing 2.5 mg/Ldicamba) with the use of an intermediate-incubation step (Table 4).Calli grown on BCI-2,4-D (barley callus induction medium [Wan andLemaux, 1994] containing 2.5 mg/L 2,4-D) displayed a shoot regenerationfrequency that was increased approximately 2.3-fold to 3.4-fold inresponse to the intermediate-incubation step. However, plantletsregenerated directly on RM grew faster than plantlets grown with anintermediate-incubation step.

[0121] Five independent transgenic lines at the fourth to sixth round ofselection were tested for green shoot regeneration with or without anintermediate-incubation step (Table 6). The transgenic lines wereobtained on selection medium (BCI-DM plus 5 mg/L bialaphos) thentransferred onto FHG (+4 mg/L bialaphos) with or without an intermediatestep. After 3-4 weeks, numbers of green spots were counted andregenerative tissues were transferred onto fresh FHG medium (+4 mg/Lbialaphos). After an additional three weeks, numbers of green shootswere counted. The regenerability of green shoots varied depending uponthe transgenic line; however, the frequency of green shoot regenerationfrom transgenic calli cultured with an intermediate-incubation stepincreased 2.8- to 11.4-fold (Table 6). Only line GPGlbGUS-13 line didnot produce any green plants, even with an intermediate-incubation step.

[0122] Discussion

[0123] In this study, two different media, DBC2 and DBC3, were used foran intermediate-incubation step to improve the regenerability oftransgenic and nontransgenic callus tissues of Golden Promise. Nosignificant difference was detected among treatments in terms of numbersof transgenic and nontransgenic calli producing green sectors (Tables 4and 6). However, transfer of tissue onto DBC2 or DBC3 induced theformation of multiple green structures, ultimately resulting in agreater number of plants, from each piece.

[0124] Calli grown on callus-induction-medium containing auxin alone(either 2,4-D or dicamba) produce green sectors or green structures fromonly small areas of each callus culture. In many cases, these greensectors do not generate plantlets on RM, possibly due to insufficientnumbers of cells being generated on RM to give rise to entire plantlets.If An intermediate incubation step is used, the number of green sectorsor structures that generate plantlets is increased. The use of 2,4-D incombination with BAP in the intermediate step might improve regenerationby allowing proliferation of green, totipotent cells capable ofproducing plants.

[0125] Nontransgenic barley callus grown on callus-induction mediumcontaining 2,4-D or dicamba alone and transgenic callus selected on CIMcontaining dicamba and bialaphos produce multiple shoot meristem-likestructures when subsequently transferred to intermediate incubationmedium containing BAP, 2,4-D, and copper (50×) under dim lightconditions (Tables 4 and 6). These meristem-like structures subsequentlyproduce multiple shoots. In contrast, medium containing BAP aloneproduces only one or a few shoots per green sector. Thus, an IIMcontaining an appropriate auxin, BAP, and copper to treat calluspromotes the production of multiple green meristem-like structures andresultant plantlets.

[0126] No significant difference in regenerability between DBC2 and DBC3(Tables 4 and 6) is observed; rather, the callus structure itselfdetermined the outcome. In general, DBC2 medium is more appropriate forcallus with smaller-sized green sectors than DBC3 medium. DBC2 mediuminhibits the growth of shoots, but green sectors or green structures canbe maintained and proliferated on this medium for a long period of timeuntil they have achieved a size appropriate for regeneration. Greentissues of Golden Promise, Galena, Harrington, and Salome, for example,can be maintained for more than 10 months (more than 4-6 months forMorex). These tissues produce multiple green shoots with a range of 9-17shoots per piece of green tissue 4-6 mm in size. When germinatingtissues were broken into 3-4 pieces after 3-4 weeks on RM andtransferred to fresh medium, an even greater number of shoots wereproduced from the small embryogenic structures in which no shoots hadyet formed.

[0127] Although the use of the intermediate-incubation step increasedregenerability, there were still transformation events which were notregenerable. For example, the GPGlbGUS-13 transgenic line did notproduce any green plants, possibly due to either transformation of asingle original nonregenerable cell or to the early loss ofregenerability during culturing of the callus. The use of anintermediate-incubation step as early as possible during theregeneration procedure also reduced the incidence of albinism. Byapplying this intermediate-incubation step at earlier selection stages,we obtained green, transgenic plants from a recalcitrant commercialcultivar called Galena, a result that was unachievable using publishedprocedures.

[0128] Compared to earlier methods (Wan and Lemaux, 1994), the use of anintermediate-incubation step increased the frequency of shootregeneration about 2.3-fold to about 11.4-fold for the nontransgenic andtransgenic calli of Golden Promise and improved the culturability andregenerability of other recalcitrant commercially important genotypes,such as the North American malting cultivars Harrington and Morex (seeTable 5). TABLE 4 Regeneration of Nontransgenic Callus Tissues of GoldenPromise Inter- Calli with Green Shoots Maintenance mediate RegenerationGreen Spots per per Callus Medium Step Medium Calli Tested Piece BCI-DMFHG FHG 4.8 ± 3.6/10 0.35 ± 0.13 (100%) DBC2 FHG 5.5 ± 3.0/10 1.95 ±0.26 (557%) DBC3 FHG 7.3 ± 1.0/10 2.23 ± 0.29 (637%) BCI-2,4-D FHG FHG7.5 ± 1.7/10 1.15 ± 0.30 (100%) DBC2 FHG 7.8 ± 2.1/10 3.88 ± 1.36 (337%)DBC3 FHG 8.5 ± 0.6/10  2.6 ± 0.52 229%

[0129] TABLE 5 Regeneration of Nontransgenic Callus Tissues of MorexMainten- Inter- Calli with Shoots per ance mediate Regeneration GreenSpots per Callus Piece Medium Step Medium Calli Tested Green AlbinoBCI-2,4-D FHG FHG 3.3/7 0 0 DBC2 FHG 3.7/7 0.29 2.4

[0130] TABLE 6 Regeneration of Transgenic Callus Tissues of GoldenPromise Inter- Regen- No. of Calli with No. of Green Transgenic mediateeration Green Sports/No. Shoots Per Line Step Medium of Calli TestedCallus GPGlbGUS-6 FHG FHG  6/10 0.1 (100%) DBC2^(a) FHG  6/10 0.7 (700%)DBC3^(b) FHG  7/10 0.4 (400%) GPGlbGUS-7 FHG FHG 6/7 0.43 (100%) DBC2FHG 6/7 1.57 (365%) DBC3 FHG 4/7 3.29 (765%) GPGlbGUS-13 FHG FHG  0/100.0 (0%) DBC2 FHG  0/10 0.0 (0%) DBC3 FHG  0/10 0.0 (0%) GPdGGUS-5 FHGFHG 10/10 1.0 (100%) DBC2 FHG 10/10 4.9 (490%) DBC3 FHG  9/10 11.4(1140%) GPdGGUS-8 FGH FHG 6/7 0.57 (100%) DBC2 FHG 5/7 2.14 (375%) DBC3FHG 3/7 1.57 275%

Example 3

[0131] Reduction of Genotype Limitation and Albinism: Transformation ofBarley Genotype Golden Promise and the Recalcitrant Barley GenotypeGalena

[0132] Materials and Methods

[0133] Plant Material. Donor plants for immature embryos were grown insoil under controlled conditions in growth chambers as described (Wanand Lemaux, 1994; Lemaux et al., 1996) or in the greenhouse, as noted(immature embryos grown in the growth chambers are preferred for greentissue culture, although it is not necessary to use greenhouse-grownplant material).

[0134] The greenhouse had supplementary lighting providing a 14-hphotoperiod with temperatures of 15 to 18° C. Supplemental 1000-wattmetal-halide lights were engaged when the light level in the greenhousewas less than 1000 μE m⁻² s⁻¹. Roof shades covered the roof when outsidelight levels rose above 7000 μE m⁻² s⁻¹.

[0135] Callus induction and green embryogenic tissue production.Immature zygotic embryos about 1.5 to 2.5 mm in size were dissected andisolated intact under a stereo dissecting microscope from seeds thatwere surface-sterilized for 10 min in 20% (v/v) bleach (5.25% sodiumhypochlorite) followed by three washes in sterile water. The embryoswere placed scutellum-side down on CIM.

[0136] Six different CIMs were used to test callus-induction frequenciesand callus quality. The CIMs had, respectively, different concentrationsof: 2,4-D (1.0 and 2.5 mg/L), BAP (0.01, 0.1 and 0.5 mg/L), and cupricsulfate (CuSO₄; 0.-1 and 5.0 FM) as shown in Table 7.

[0137] DBC1 medium, which is CIM with 2.5 mg/L 2,4-D, 0.01 mg/L BAP, and5.0 μM CuSO₄, was used for the initial callus-induction period withGolden Promise. DBC2 medium was used for the initial callus-inductionperiod with Galena and Salome.

[0138] Five to seven days after callus initiation, germinating shootsand roots were removed from the callusing scutellum by manual excision.After 3-4 weeks initial incubation in the dark at 24±1° C., embryogeniccallus from the scutellum was cut into small pieces (about 3-4 mm),transferred to fresh DBC2 medium (Golden Promise and Galena), and grownunder dim light conditions (approximately 10 to 20 μE m⁻²s⁻¹, 16h-light). After an additional three weeks (at the second transfer),green callusing sectors were selected, broken into two to three pieces(each about 3-4 mm-in size) and transferred to fresh DBC2 medium.

[0139] Green regenerative tissues from Golden Promise and Salome weremaintained on DBC2 medium, subculturing at three to four-week intervals.

[0140] DBC3 medium was used from the second transfer for Galena andsubculturing took place at three to four-week intervals.

[0141] Plant regeneration. Seven pieces of four-month-old greenregenerative tissue (about 4-6 mm) were plated on solid RM and exposedto a light intensity of approximately 30 to 50 μE m⁻²s⁻¹. After 25-days,the numbers of green tissues that produced shoots and the numbers ofshoots per piece of green tissue were counted. A single base of greentissue with more than one leaf was considered as one shoot.

[0142] Regenerated shoots were transferred to rooting medium (CI mediumwithout hormones) in Magenta® boxes (Magenta Corporation, Chicago,Ill.). When the shoot reached the top of the box (approximately 3-4weeks), plantlets were transferred to 6-inch pots containing Supersoil™(R. McClellan, S. San Francisco, Calif.), gradually acclimatized, andgrown to maturity in the greenhouse.

[0143] Plasmid. Plasmid pAHC25 includes the uidA (gus) reporter gene anda selectable gene, bar, each under control of the maize ubiquitin Ubilpromoter and intron 1 and terminated by nos (Christensen and Quail,1996).

[0144] DNA particle bombardment. Intact barley embryos weresurface-sterilized, placed scutellum-side down, and grown on CIM, eithersupplemented with 2.5 mg/L 2,4-D and 5.0 μM CuSO₄ (DC medium) orsupplemented with 2.5 mg/L 2,4-D and 0.1 μM CuSO₄ (D medium).

[0145] One day after excision of Galena embryos at 24±1° C. in the dark,the embryos were transferred scutellum-side up for osmotic pretreatmenton CIM containing no maltose but including 0.2 M mannitol and 0.2 Msorbitol. Four hours after treatment with the osmoticum, the embryoswere bombarded as described (Lemaux et al. 1996). Briefly, this involvedthe coating of 1 μm gold particles (Analytical Scientific Instruments,Alameda, Calif.) with plasmid DNA followed by bombardment using aPDS-1000 He biolistic device (Bio-Rad, Inc., Hercules, Calif.) at 900psi 16-18 hours after bombardment, the embryos were placedscutellum-side down on DC medium (no bialaphos) and grown at 24+1° C. inthe dark for 10-14 days.

[0146] Selection and regeneration of transformed tissue. Following aninitial 10- to 14-day culturing period, each callusing embryo was brokeninto two or three pieces (approximately 4-5 mm each), depending oncallus size, transferred to DBC2 medium supplemented with 4 mg/Lbialaphos, and incubated in the dark. Two weeks after the secondtransfer (first-round selection), callus was transferred to new DBC2medium containing 4 mg/L bialaphos, and 7 to 14 days later, calli weremoved to dim light conditions (about 10 μE m⁻²s⁻¹, 16 h-light). Throughthe fourth transfer, calli were maintained on the same medium. At thefifth transfer, calli were moved to DBC3 medium supplemented with 4 mg/Lbialaphos. Cultures were subcultured at two-week intervals on DBC3medium with 4 mg/L bialaphos until formation of green structuresoccurred, at which time they were plated on solid RM containing 3 mg/Lbialaphos for regeneration and exposed to higher intensity light(approximately 30-50 μE m⁻² s⁻¹) After 3-4 weeks on RM, regeneratedshoots were transferred to Magenta® boxes containing rooting medium (CImedium containing 0.1 μM copper without hormones) supplemented with 2-3mg/L bialaphos. When the shoots reached the top of the box, plantletswere treated as described above.

[0147] Histochemical GUS assay. GUS activity was assayed histochemicallyas described (Jefferson et al., 1987).

[0148] PCR assay. Polymerase chain reaction (PCR) analysis was carriedout using genomic DNA extracted from calli or leaves. Two sets ofprimers were used for confirming the presence of the bar gene, Bar5F andBarlR (Lemaux et al., 1996). Another set of primers was used forconfirming the presence of the gus gene, uidA1 and uidA2R (Cho et al.,1996). Amplifications were performed in a 25 μL reaction volumecontaining 10×PCR buffer, 25 mM MgCl₂, 2.5 mM dNTPs, 20 μM each primer,with 0.25 μL Taq DNA polymerase (Promega). Cycling was controlled by athermal cycler programmed with the following conditions: 1 mindenaturation step at 94° C.; 10 cycles at: 94° C. for 45 sec, 60° C. for0.1-0.5 min/cycle, 72° C. for 1 min; and −26 cycles at: 94° C. for 45sec, 55° C. for 1 min, 72° C. for 1 min. For the final cycle, theduration of the extension step was 7 min at 72° C. 25 μL of the PCRproduct with loading dye was electrophoresed on a 0.8% agarose gel withethiaium bromide and detected by UV light.

[0149] DNA hybridization analysis. Genomic DNA isolated from leaf tissueof a nontransformed control plant and T₀ and T₁, plants of transgeniclines was digested with XbaI and either SacI or PstI. Digestion withXbaI and Sad releases an intact 1.8 kb uidA (gus) fragment; digestionwith XbaI and PstI releases an intact 0.6 kb bar fragment. For gelelectrophoresis, each lane was loaded with 10 μg of each digest. AfterSouthern transfer, the resulting blot was hybridized with a ³²P-labeleduidA or bar probe.

[0150] Results

[0151] Initial callus induction and growth. The initial callus-inductionfrequency was determined using CIM of different compositions. Tenimmature embryos from the barley genotypes Golden Promise and Galenawere transferred to each CIM. Each CIM contained the following levels ofhormones and copper: D, 2.5 mg/L 2,4-D and 0.1 μM CuSO₄; DC, 2.5 mg/L2,4-D and 5.0 μM CuSO₄; DB, 2.5 mg/L 2,4-D, 0.1 mg/L BAP 0.1 μM CuSO₄;DBC1, 2.5 mg/L 2,4-D, 0.01 mg/L BAP and 5.0 μM CuSO₄; DBC2, 2.5 mg/L2,4-D, 0.1 mg/L BAP and 5.0 μM CuSO₄; DBC3, 1.0 mg/L 2,4-D, 0.5 mg/L BAPand 5.0 μM, CuSO₄. Callus quality was assessed microscopically andscored on a scale with +++++ designating highest quality and +designating lowest quality. Values were measured three weeks afterinitial callus induction and represent means of three replicates foreach treatment. Golden Promise had a high frequency of callus inductionregardless of the CIM composition (>87%, Table 7). Galena had a highfrequency of callus induction (>90%) on CIM without BAP. Tissue qualitywas poor on CIM containing BAP three weeks after induction but improvedafter two to three transfers on this medium. Only a fraction ofscutellar tissues on Galena immature embryos formed callus, while mostof the scutellar surface on Golden Promise immature embryos formedhigh-quality callus.

[0152] Galena had a similar or slightly higher initial callus growthrate compared to Golden Promise when grown on D medium (CIM containing2,4-D alone) or DC medium (CIM containing 2,4-D and elevated levels ofcopper) (Table 7). Increasing the level of copper to 5.0 μM (50×) didnot change the callus-induction frequency or the initial callus growthrate in either genotype, but callus quality improved, especially inGolden Promise. Compared to Galena, Golden Promise immature embryosproduced callus with a larger number of distinct embryogenic structures.

[0153] The addition of BAP to the CIM reduced the callus-inductionfrequency and inhibited callus growth for both genotypes but producedhigher quality callus that was shiny, compact, and contained highlyregenerative structures with multiple shoot meristems (Table 7). Galenarequired a higher level of BAP (0.1 mg/L) than Golden Promise (0.01 mg/LBAP) to obtain callus of high quality (Table 7). The higher level ofcopper (50×) in combination with BAP resulted in more regenerativestructures from callus having a slightly brownish color. When DBC3medium, which contains a higher level of BAP (0.5 mg/L) and a lowerlevel of 2,4-D (1.0 mg/L), was used for initial callus induction, a highrate of embryo germination and production of poor quality callus with aslow growth rate occurred (Table 7).

[0154] Production and maintenance of green regenerative tissues. Greenembryogenic structures were observed 5-20 days after exposure of 3-4week-old callus to dim light. A higher percentage of green sectors wasproduced by Golden Promise callus than Galena callus tissue. Once acallus having the appropriate morphology under dim light conditions wasidentified (green, shiny, nodular, compact), the sectors could be easilyseparated from the remaining callus and maintained on either DBC2 orDBC3 medium. Approximately 6-8 weeks post-initiation on DBC2 medium,Golden Promise tissue contained a few green shoots with multiple shootmeristem-like structures, but most tissues were green, shiny, nodularand compact. For Galena, however, DBC3 medium was optimal formaintaining green regenerative tissues. On DBC3 medium, Golden Promisetissues were softer and produced multiple shoot meristems; germinationof some shoots was induced in response to a higher level of BAP. Galenatissues produced multiple shoot meristems and were more compact on DBC3medium than Golden Promise tissues.

[0155] Thus, Galena requires a-higher level of BAP (0.5 mg/L) thanGolden Promise (0.1 mg/L) for callus induction and maintenance ofhigh-quality green regenerative tissues. It should be noted that inthese experiments, the callus-induction media used do not contain a highlevel of copper. Callus morphology was very good with 0.1 mg/L BAP, butthe growth rate was very slow. When 50× copper was added, it seemed tospeed up the growth rate. A higher level of BAP in callus-inductionmedium containing 50× copper was needed for optimal growth of tissuescompared with callus-induction medium containing 1× copper.

[0156] Fertile plant regeneration from green regenerative tissues. Sevenpieces of green embryogenic tissues 4 to 6 mm in size from each genotypewere transferred to RM (FHG medium), and after 25 days the number ofregenerated shoots were counted. Each piece yielded multiple greenshoots. After 2-3 weeks on RM, green structures (4-6 mm in size) fromboth genotypes produced approximately 9-17 green shoots per piece oneither RM (Table 8) or hormone-free rooting medium. When germinatingtissues were broken into pieces after 3-4 weeks in culture on RM andtransferred to fresh medium, an even larger number of shoots wereproduced from the small green structures. All four-month-old greenstructures tested for regeneration produced multiple green shoots; noalbino plants were observed (Table 8). Regenerated shoots weretransferred to rooting medium in Magenta® boxes and rooted plants weretransferred to soil and grown to maturity in the greenhouse.

[0157] Transformation of Galena. The in vitro culture system describedabove results in multiple green shoots from immature embryo-derivedcallus, thus providing the basis for successful transformation of therecalcitrant commercial genotype, Galena.

[0158] For transformation, the scutella of immature embryos of Galenawere bombarded with subsequent culturing of the embryos on DC medium inthe absence of selection. From the second transfer on, calli weremaintained on selection medium; in the middle of the third round oftransfer, calli-were moved to dim light. Media containing higher levelsof BAP, lower levels of 2,4-D, and 50× copper (DBC3 medium) were usedfor selection and maintenance from the fifth transfer on. In general,bialaphos-resistant calli with green sectors were observed at the fourthto fifth transfer. Calli with green sectors were maintained andproliferated until the green sectors formed fully developed regenerativestructures. In most cases, when green sectors developed in fast-growingcallus, fully developed green regenerative structures could be obtained.

[0159] For Galena, embryo size was very important for callus induction.Embryos smaller than about 1.2 mm resulted in very poor callus induction(less than 20 percent). Immature embryos about 1.5 mm to 2.0 mm in sizehad the highest callus induction frequency (>90 percent).

[0160] This method of generating green structures that yield, multiplegreen shoots was used to improve the regenerability of transgenic calliselected on CIM containing either dicamba or 2,4-D. Green sectors wereregenerated under selection and the plantlets were transferred to soilapproximately three weeks after transfer to rooting medium. Using thistransformation protocol, we obtained six independent Galena linestransformed with pAHC25. Three lines produced green sectors, wereregenerable, and produced multiple green shoots. T₀ and T₁, plantscontained DNA sequences that hybridized to bar and uidA and functionallyexpressed the uidA (GUS) reporter gene and the herbicide resistance genebar as judged by resistance to Basta™.

[0161] Discussion

[0162] We have developed a very efficient, reproducible system forproducing highly regenerative callus that gives rise to multiple greenshoots over long periods of time (Table 8), eliminating the problem ofalbinism. This system can be successfully used to transform andregenerate previously recalcitrant genotypes.

[0163] First, we have optimized phytohormone treatment during callusinitiation and proliferation. Immature embryos from Galena requiredhigher levels of BAP than Golden Promise in order to produce highquality, green, regenerative tissue, perhaps due to differences betweenthe two genotypes in endogenous levels of phytohormones. The addition ofBAP to CIM containing 2,4-D decreased the growth rate of the immatureembryo-derived callus from both genotypes but improved its quality andregenerability (Table 7). It is possible that the lack of albinism inthis study was at least partially attributable to the use of BAP.

[0164] Recently, in vitro culture systems utilizing 2,4-D and BAP weredeveloped for the differentiation of multiple shoots from excised shootapical meristems from maize (Zhong et al., 1992) and oat (Zhang et al.,1996). We found that the sectors of regenerative barley calli of GoldenPromise and Galena that were grown on CIM containing 2,4-D or dicambaproduced multiple shoot meristem-like structures when subsequentlytransferred to an intermediate-incubation medium containing 2,4-D andBAP under dim light conditions. The use of 2,4-D in combination with BAPprovided more prolonged regenerability and was more applicable to othergenotypes than dicamba in combination with BAP.

[0165] Other changes in culture conditions significantly improved invitro manipulation. Compared to D medium, DC medium, which includesincreased levels of copper (5.0 μM, a 50-fold increase from MS medium),improved callus quality (Table 7) without changing callus-inductionfrequencies or the initial callus growth rate. This provided higherquality material from the initial step of selection that led toincreased regenerablity in transformed tissues. In addition, the use ofDBC2 medium at the second transfer of Galena resulted in higher qualitytissue that produced multiple shoot meristem-like structures.

[0166] These results were consistent with studies indicating that 50 μMcopper (500×) is optimal for regenerability of the barley varietyHector, while 5.0 μM is optimal for regenerability for the barleyvariety Excel (Dahleen, 1996). Similar results were reported for wheat,wherein regeneration was reportedly higher on medium containing 10 μMCuSO₄ (100×) than on MS (0.1 μM Cu²⁺) (Purmhauser, 1991). In yet anotherstudy, an increased copper level resulted in more somatic embryoids fromanthers of tetraploid wheat (Ghaemi et al., 1994).

[0167] The exposure of tissue to light early in the selection processalso likely reduced the incidence of albinism, perhaps by inducingchlorophyll biosynthetic enzymes (Holtorf et al., 1995). The presence ofgreen, regenerative sectors assures that green plants will be generated,thus decreasing or eliminating the regeneration of albino plants asobserved in Wan and Lemaux (1994).

[0168] Shiny, compact, slightly brown-colored calli with highlyregenerative structures were obtained 2-3 weeks after incubating embryosin the dark on CIM. For both genotypes, the calli were transferred tofresh medium containing BAP, 2,4-D and copper, and green embryogenicstructures were formed 5-14 days after exposure to dim light. Allfour-month-old regenerative structures of both Golden Promise and Galenaregenerated multiple green shoots (approximately 11-17 green shoots percallus piece, Table 8) and no albino plants. By contrast, four-month-oldcallus of Golden Promise and Galena maintained on CIM containing either2.5 mg/L 2,4-D or dicamba alone did not produce green shoots (Example1); even two-month-old callus of Golden Promise maintained on CIMcontaining 2,4-D or dicamba alone produced only 0.35 and 1.15 greenshoots per callus piece, respectively (Example 2). These regenerativestructures could be maintained on 2,4-D, BAP and copper for more thanten months in this state and could be regenerated to give multiplefertile green plants with both genotypes. The morphology of the greentissues generated by our protocol was similar to that of the multiplegreen meristematic domes differentiated from shoot apical meristemsfollowing culture on 2,4-D and BAP, but was more compact, possibly dueto the inherent differences in the tissue source or to the use of higherconcentrations of 2,4-D.

[0169] It has been reported that callus quality and callus-inductionfrequency depend on the selection of appropriately sized embryos andoptimization of the physiological state of the donor plant (Dale andDambrogio, 1979; Goldenstein and Kronstadt, 1986; Lürz and Lörz, 1987;Wan and Lemaux, 1994). However, the green regenerative tissues producedusing our protocol can be obtained from a wider range of embryo sizesand from plants grown in either the growth chamber or the greenhouse;once green tissues are generated from any source, they can beproliferated as described. Small embryo size (<1.0 mm) was better incallus induction for Golden Promise, Morex, and Salome, but Galenarequired a larger size (1.5 to 2.0 mm) of embryos for a highercallus-induction frequency.

[0170] Many barley genotypes have a very low callus-induction frequency(Lürz and Lörz, 1987; Dahleen, 1996) and the appearance of albino plantsand low regenerability occurs within 2.5 months after callus induction(Bregitzer et al., 1995). These traits limit the applicability ofbarley-transformation procedures for many modem commercial genotypes.

[0171] Previous efforts to transform the commercial varieties MoravianIII and Galena produced large numbers of independently transformedlines, but yielded only albino plants upon regeneration. Changing thelevel of selection (to 1 mg/L bialaphos) or shortening the time ofselection (from >5 rounds to 3 rounds) led to the regeneration of greenplants that were found to be nontransformed.

[0172] The methods disclosed herein obviate the problems of albinismencountered with prolonged culture periods; in this study Galena andGolden Promise could be regenerated to give fertile green plants formore than 10 months. In addition, the use of either DBC2 or DBC3 in anintermediate step in regeneration greatly improves the frequency ofshoot regeneration of transgenic and nontransgenic callus from GoldenPromise initiated on 2,4-D or dicamba (Example 2).

[0173] Changes in particle bombardment, selection and culturingconditions, among others, also contributed to our transformation successwith the previously recalcitrant genotype, Galena. Bombardments werepreviously carried out at 1100 psi, resulting in a reduction incallus-induction frequency in Galena, although Golden Promise wasunaffected in its frequency. It is possible that lowering the rupturepressure and hence the speed of the microprojectiles lessened damage tothe target tissue. In addition, selection of Galena was initiated twoweeks post-bombardment rather than one day post-bombardment (Wan andLemaux, 1994) in order to promote better callus induction and to allowfor vigorous cell divisions of transformed cells without the adverseeffects of the dead or dying cells in close proximity resulting fromselection. Also, callusing embryos were broken into large pieces (4-5mm) to avoid potential negative effects of wounding on transformedcells.

[0174] The approaches detailed herein can be used successfully totransform other recalcitrant commercial genotypes, such as the NorthAmerican barley cultivars, Harrington, a two-row variety, and Morex, asix-row variety. Using these methods, Harrington and Morex produce greenregenerative structures that yield multiple shoot meristems.

[0175] In addition, the ability to maintain green regenerativestructures for long periods of time in culture permits the use of thesestructures as target tissues for transformation of cultivars prone toalbinism, thus eliminating the need for maintaining donor plants anddecreasing problems with albinism and poor regenerability, as well asreducing the induced mutation frequency and the resultant somaclonalvariation. TABLE 7 Callus-Induction-Frequency, Initial Callus GrowthRate and Callus Morpholocry of Golden Promise (GP) and Galena onDifferent Callus-Induction Media Callus- Initial Callus- InductionInduction Growth Rate Callus Genotype Medium Frequency (%)(mg/day/embryo) Morphology GP D 100  12.8 ++ DC 100  10.6 ++++ DB 97 7.0+++(+) DBC1 100  11.5 +++++ DBC2 100  9.1 +++(+) DBC3 87 6.5 ++ Galena D97 15.0 + DC 90 13.6 +(+) DB 67 5.7 ++(+) DBC1 80 8.3 ++ DBC2 47 5.8 +++DBC3 47 3.1 ++

[0176] TABLE 8 Number of Shoots Regenerated from Green EmbryogenicTissues of Golden Promise and Galena # Regenerated Medium forShoots/Green Tissue^(c) Genotypes Maintenance Green Albino GoldenPromise DBC2^(a) 17.0 0 Galena DBC2 13.0 0 Galena DBC3^(b) 14.4 0

Example 4

[0177] Use of Green Regenerative Tissues of Barley as TransformationTargets

[0178] Materials and Methods

[0179] Plasmids. Plasmids pAHC20 and pAHC25 are described above. pAHC15contains the GUS reporter gene expression cassette of pAHC25(Christensen and Quail, 1996).

[0180] pUbiINPTII-1 was constructed by inserting the neomycinphosphotransferase (NPTII) coding sequence from pCaMVNEO (Fromm et al.,1986) into the BamHI site of pAHC17 which contains the maize ubiquitinUbil promoter, Ubil intron 1, and the nos 3′ terminator (Christensen andQuail, 1996).

[0181] Preparation of green regenerative tissues for DNA particlebombardment. Immature zygotic embryos were surface-sterilized, placedscutellum-side down on DBC2 medium, and incubated at 24±1° C.Regenerative tissues were maintained for 3-4 weeks, then cut into smallpieces (about 3 to 5 mm), transferred to fresh DBC2 medium, and grownunder dim light conditions. After an additional three weeks, greencallusing sectors were broken into pieces (about 3 to 5 mm) andtransferred to fresh DBC2 medium. Green regenerative tissues weremaintained on DBC2 medium with subculturing at 3- to 4-week intervals.CIM containing 2.5 mg/L 2,4-D, 0.1 mg/L BAP and 5.0 μM CuSO₄ (i.e., DBC2medium) was used for the induction of green regenerative tissues fromthe other genotypes.

[0182] For bombardment, green tissues (about 3 to 5 mm, four-months old)of Golden Promise and Galena were placed in the dark at 24±1° C. for oneday, then transferred to DBC2 medium containing 0.2 M mannitol and 0.2 Msorbitol-. Four hours after treatment with the osmoticum, green tissueswere bombarded as described (Lemaux et al. 1996) with gold particles(Analytical Scientific Instruments, Alameda, Calif.) coated with pAHC25,a mixture of pAHC20 and pAHC15, or a mixture of pUbiINPTII-1 and pAHC15at 900 or 1100 psi. At 16-18 hours after bombardment, the green tissueswere transferred to DBC2 medium without osmoticum and grown at 24±1° C.under dim light conditions (about 10 μE m⁻² s⁻, 16 h-light).

[0183] Selection and Regeneration of Transformed Tissue. Following aninitial 3- to 4-week culturing period on nonselective medium, each pieceof green tissue was broken into 1 to 2 pieces (about 4 mm to 5 mm,depending on size of original tissue piece) and transferred to DBC2medium (Golden Promise or Galena) or DBC3 (Galena) medium supplementedwith 4 to 6 mg/L bialaphos for bar selection or 40 to 50 mg/L geneticin(G418) for nptII selection. Green tissues were selected on DBC2 or DBC3medium and 4 mm to 5 mm tissues subcultured at 3- to 4-week intervals.Putative green tissue transformants, identified by their fast-growthcharacter on the selective medium, were transferred to Magenta® boxescontaining rooting medium that was supplemented either with 4 mg/Lbialaphos for bar selection or without selective agent for regenerationof nptII transformants. When shoots reached the top of the box,plantlets were transferred to 6-inch pots containing Supersoil (R.McClellan, S. San Francisco, Calif.), gradually acclimatized, and grownto maturity in the greenhouse.

[0184] Results and Discussion

[0185] Various targets have been used for barley transformation,including immature zygotic embryos (Wan and Lemaux, 1994; Hagic, et al.,1995), young callus (Wan and Lemaux, 1994), microspore-derived embryos(Wan and Lemaux, 1994), microspores (Jähne et al., 1994) and protoplasts(Funatsuki et al, 1995; Salmenkallio-Marttila et al., 1995). Immaturezygotic embryos are currently the most widely used and reliable targettissue for barley transformation. However, immature embryos from mostcommercially important barley genotypes have low callus-inductionresponse rates (Lürz and Lörz, 1987; Dahleen, 1996). Moreover, invitro-derived tissue culture material is limited in its ability to yieldgreen plants for prolonged periods (Bregitzer et al., 1995). Prolongedculturing periods and/or selection stress required during thetransformation process result in a large proportion of albino(chlorophyll-deficient) plants (Foroughi-Wehr et al., 1982; Wan andLemaux, 1994; Bregitzer et al., 1995). In addition, the use of immatureembryos and microspores as target tissues requires the year-roundmaintenance of donor plants grown under defined growth conditions.

[0186] We have established a reproducible barley transformation systembased on microprojectile bombardment of green tissues that utilizes anin vitro culture system for the production of multiple green shoots fromcallus derived from immature scutellar tissue. Selection commenced 3 to4 weeks after bombardment to allow transformed cells to proliferate inthe absence of dead or dying cells resulting from selection or wounding.From the second transfer, selection was started using DBC2 medium orDBC3 medium that was supplemented either with bialaphos for barselection or G418 (geneticin) for nptII selection. Putativetransformants identified after 3 to 4 rounds of selection weretransferred to rooting medium supplemented with bialaphos.

[0187] Using this transformation protocol we have obtained one confirmedGolden Promise line transformed with pAHC20 plus pAHC15 followingselection with bialaphos, plus one putative transformed Galena line withpUbiINPTII-1 plus pARC15 after G418 selection. Both lines wereregenerable, producing green shoots and plants. Transformation wasconfirmed by PCR analysis.

[0188] This protocol greatly reduces problems with albinism and poorregeneration observed previously (Wan and Lemaux, 1994; Foroughi-Wehr etal., 1982; Bregitzer et al, 1995; Koprek et al, 1996; etc.) and can alsobe applied to other recalcitrant barley cultivars such as Harrington andMorex.

Example 5

[0189] Callus Morphology of Wheat on Different Callus Induction Media

[0190] The tissue culture protocols described above for use with barleyare also useful for a variety of other plant species, including variousmonocot species.

[0191] For example, we have shown that the wheat variety Bobwhite alsoshows improved initial callus induction and callus morphology whentested on CIM containing high levels of copper and BAP. In experimentsconducted as in Example 1 above (except as noted), immature wholeembryos (1-2 mm) of Bobwhite were tested on six different CIMs, eachincluding MS medium supplemented with 30 g/L maltose, 0.5 mg/Lthiamine-HCl, 150 mg/L asparagine, and solidified with 2.5 g/L Phytagel(pH 5.85), and supplemented with copper and phytohormones as follows:(1) WD: 2.0 mg/L 2,4-D and 0.1 μM CuSO₄. (2) WDC: 2.0 mg/L 2,4-D and 5.0μM CuSO₄. (3) WDB: 2.0 mg/L 2,4-D, 0.1 mg/L BAP, and 0.1 μM CuSO₄. (4)WDBC1: 2.0 mg/L 2,4-D, 0.01 mg/L BAP, and 5.0 μM CuSO₄. (5) WDBC2: 2.0mg/L 2,4-D, 0.1 mg/L BAP, and 5.0 μM CuSO₄. (4) WDBC3: 2.0 mg/L 2,4-D,0.5 mg/L BAP, and 5.0 μM CuSO₄.

[0192] The shoot apex was removed seven days after callus induction. Themorphology of the callus induced on the media is shown in Table 9.

[0193] Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims. TABLE 9 Callus Morpholocry ofBobwhite Wheat on Different, Callus-Induction Media Callus-InductionMedium Callus Morphology^(a) WB ++ WDC ++(+) WDB +++ WDBC1 +++ WDBC2++++ WDBC3 +++++

REFERENCES

[0194] Ausubel et al., eds. (1992, with periodic updates) CurrentProtocols in Molecular Biology, Greene Publishing andWiley-Interscience, New York.

[0195] Baillie et al. (1993) Can. J. Plant Sci. 73: 171-174.

[0196] Bhaskaran and Smith (1990) Crop Sci. 30:1328-1336.

[0197] Bregitzer (1992) Crop Sci. 32:1108-1112.

[0198] Bregitzer et al (1995) Plant Cell Tiss. Org. Cult. 43:229-235.

[0199] Breimann (1985) Plant Cell Rep. 4:161-163.

[0200] Christensen and Quail (1996) Transgenic Res. 5:1-6.

[0201] Dahleen (1996) Plant Cell Tiss. Org. Cult. 43:267-269.

[0202] Dale and Dambrogio (1979) Z. Pflanzenphysiol. 94:65-77.

[0203] De Block et al. (1987) EMBO J. 6:2513-2518.

[0204] Dmitrieva (1985) “Hormones, Dedifferentiation and Control ofProliferation in Cell and Protoplast Cultures,” in: Butenko, ed., PlantCell Culture, Biology Series, MIR Publishers, Moscow, pp. 35-50.

[0205] Fletcher (1969) Planta 89:1-8.

[0206] Foroughi-Wehr et al. (1982) Theor. Appl. Genet. 62: 233-239.

[0207] Fromm et al. (1986) Nature 319:791-793.

[0208] Fromm et al. (1989) Plant Cell 1:977.

[0209] Funatuski et al. (1995) Theor. Appl. Genet. 91:707-712.

[0210] Gelvin et al. (1990) Plant Molecular Biology Manual, KluwerAcademic Publishers.

[0211] Ghaemi et al. (1994) Plant Cell Tiss. Org. Cult. 36: 355-359.

[0212] Goldenstein and Kronstadt (1986) Theor. Appl Genet. 71:631-636.

[0213] Gordon-Karmm et al. (1990) Plant Cell 2:603.

[0214] Griffin and Dibble (1995) Plant Cell Rep. 14:721-724.

[0215] Hagio et al. (1995) Plant Cell Rep. 14:329-334.

[0216] Handel et al. (1985) Crop Sci. 25:27-31.

[0217] Holtorf et al. (1995) Proc. Natl. Acad. Sci. USA 92:3254-3258.

[0218] Hunter (1988) “Plant regeneration from microspores of barley,Hordeum vulgare,” PhD thesis, Wye College, University of London,Ashford, Kent, England.

[0219] Jahne et al. (1991) Plant Cell. Rep. 10: 1-6.

[0220] Jahne et al. (1994) Theor. Appl. Genet. 89:525-533.

[0221] Jefferson et al. (1987) EMBO LT. 6:3901-3907.

[0222] Kao et al. (1991) Plant Cell Rep. 9:595-601.

[0223] Kasha et al. (1990) “Haploids in cereal improvement: Anther andmicrospore culture,” in: Gene manipulation in plant improvement: 11,ed., Gustafson, Plenum, N.Y., pp. 213-235.

[0224] Koprek et al. (1996) Plant Sci. 119:79-91.

[0225] Kott and Kasha (1984) Can. J. Bot. 62:1245-1249.

[0226] Lemaux et al (1996) Bombardment-mediated transformation methodsfor barley, Bio-Rad US/EG Bulletin 2007.

[0227] Lewin (1994) Genes V, Oxford University Press: New York.

[0228] Liu (1994) Analysis of ABA-Regulated Expression of the Maize GLB1Gene in Tobacco Seeds and Maize Cells, Ph.D. thesis, University ofIllinois at Urbana-Champaign.

[0229] Lürz and Lörz (1987) Theor. Appl. Genet. 75:16-25.

[0230] Murakami et al. (1986) Mol. Gen. Genet. 205:42-50.

[0231] Murashige and Skoog (1962) Physiol. Plant. 15:473-497.

[0232] Pouwels et al. (1985, supp. 1987) Cloning Vectors: A LaboratoryManual.

[0233] Purnhauser (1991) Cereal Res. Comm. 19:419-423.

[0234] Rhagaran (1986) Embryogenesis in Angiosperms, Cambridge Univ.Press: Cambridge, p. 303.

[0235] Rieger et al. (1991) Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag_: New York.

[0236] Roshal et al. (1987) EMBO LT. 6:1155.

[0237] Salmenkallio-Marttila et al. (1995) Plant Cell Rep. 15:301-304.

[0238] Sambrook et al. (eds.) (1989), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y.

[0239] Thomas and Scott (1985) Plant Cell Rep. 15:301-304.

[0240] Thompson et al. (1987) EMBO J 6:2519-2523.

[0241] Tisserat (1985) “Embryogenesis, Organogenesis and PlantRegeneration,” in: Dixon, ed., Plant: Cell Culture: A PracticalApproach, Practical Approach Series, IRL Press, Oxford, Washington,D.C., pp. 79-105.

[0242] Vasil (1984) Cell Culture and Somatic Cell Genetics of Plants,Vols. I-III, Laboratory Procedures and Their Applications, AcademicPress: New York.

[0243] Wan and Lemaux (1994) Plant Physiol. 104:37-48.

[0244] Weissbach and Weissbach (1989) Methods for Plant MolecularBiology, Academic Press: New York.

[0245] Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.

[0246] Zhang et al (1996) J. Plant Physiol. 148:667-671.

[0247] Zhong et al. (1991) Plant Cell Rep. 10:453-456.

[0248] Zhong et al. (1992) Planta 187:483-489.

[0249] Ziauddin and Kasha (1990) Euphytica 48:171-176.

What is claimed is:
 1. A method of preparing green regenerative tissueof barley, suitable for transformation, comprising: incubating barleytissue on a callus-induction medium under dim light conditions for asufficient time to produce green regenerative tissue, wherein thecallus-induction medium comprises auxin at a concentration of about 0.1mg/L to about 5 mg/L and copper at a concentration of about 0.1 μM toabout 50 μM.
 2. The method of claim 1 , wherein the callus-inductionmedium further comprises cytokinin at a concentration of about 0.01 mg/Lto about 2 mg/L.
 3. The method of claim 2 , wherein the auxinconcentration is about 1 mg/L to about 2.5 mg/L and the cytokininconcentration is about 0.01 mg/L to about 0.5 mg/L.
 4. The method ofclaim 2 , wherein the auxin concentration is about 1 mg/L to about 2.5mg/L, and the cytokinin is about 0.1 mg/L to about 2 mg/L.
 5. The methodof claim 1 , wherein the auxin is selected from the group consisting of2,4-dichlorophenoxyacetic acid, dicamba, naphthaleneacetic acid,indoleacetic acid, and mixtures thereof.
 6. The method of claim 2 ,wherein the cytokinin is selected from the group consisting of6-benzylaminopurine, zeatin, zeatin riboside, kinetin, 2iP, and mixturesthereof.
 7. The method of claim 1 , wherein the barley tissue is zygoticembryo tissue.
 8. The method of claim 1 , wherein the callus-inductionmedium further comprises maltose at a concentration which is capable ofproducing callus that is competent to form regenerative tissue.
 9. Themethod of claim 1 , wherein the barley tissue includes transformedbarley cells.
 10. The method of claim 1 , wherein the green regenerativetissue is capable of being regenerated into a barley plant.
 11. Themethod of claim 1 further comprising incubating the barley tissue on anintermediate-incubation medium, wherein the intermediate-inductionmedium comprises auxin at a concentration of about 0.1 mg/L to about 5mg/L, cytokinin at a concentration of about 0.1 to about 5 mg/L andcopper at a concentration of about 0.1 to about 50 μM.
 12. The method ofclaim 11 , wherein the auxin is selected from the group consisting of2,4-dichlorophenoxyacetic acid, dicamba, naphthaleneaceetic acid,indoleacetic acid, and mixtures thereof.
 13. The method of claim 11 ,wherein the cytokinin is selected from the group consisting of6-benzylaminopurine, zeatin, zeatin riboside, kinetin, 2iP, and mixturesthereof.
 14. The method of claim 11 , wherein the intermediate-inductionmedium comprises auxin at a concentration of about 0.5 mg/L to about 2.5mg/L.
 15. The method of claim 11 , wherein the intermediate-inductionmedium comprises maltose as a sugar source.
 16. The method of claim 11 ,wherein the intermediate-induction medium comprises maltose at aconcentration which is capable of proliferating the regenerative tissue.17. The method of claim 11 , wherein the barley tissue includestransformed barley cells.
 18. The method of claim 11 , wherein theintermediate-induction medium comprises maltose at a concentration whichis capable of forming the regenerative tissue.
 19. The method of claim11 , wherein the intermediate-induction medium comprises maltose at aconcentration which is capable of forming and proliferating theregenerative tissue.
 20. The method of claim 11 , wherein the barleytissue is zygotic embryo tissue.